Inhibition of Arabidopsis O-Acetylserine(thiol)lyase A1 by Tyrosine Nitration

• Published in: The Journal of biological chemistry Vol. 286; no. 1; pp. 578 - 586
• Main Authors: Álvarez, Consolación, Lozano-Juste, Jorge, Romero, Luís C., García, Irene, Gotor, Cecilia, León, José
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.01.2011; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Arabidopsis; Arabidopsis - drug effects; Arabidopsis - enzymology; Arabidopsis - genetics; Binding Sites; Biocatalysis; Carbon-Oxygen Lyases - antagonists & inhibitors; Carbon-Oxygen Lyases - chemistry; Carbon-Oxygen Lyases - genetics; Carbon-Oxygen Lyases - metabolism; Enzyme Catalysis; Enzyme Inactivation; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - pharmacology; Enzyme Structure; Mass Spectrometry (MS); Models, Molecular; Mutation; Nitric Oxide; Oxidative Stress; Peroxynitrous Acid - metabolism; Peroxynitrous Acid - pharmacology; Plant; Plant Biology; Plant Extracts - metabolism; Protein Chemical Modification; Protein Conformation; Protein Processing, Post-Translational; Tyrosine - metabolism; Tyrosine Nitration; Enzyme Structure; Tyrosine Nitration; Plant; Oxidative Stress; Arabidopsis; Enzyme Catalysis; Protein Chemical Modification; Mass Spectrometry (MS); Nitric Oxide; Enzyme Inactivation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.147678

The last step of sulfur assimilation is catalyzed by O-acetylserine(thiol)lyase (OASTL) enzymes. OASTLs are encoded by a multigene family in the model plant Arabidopsis thaliana. Cytosolic OASA1 enzyme is the main source of OASTL activity and thus crucial for cysteine homeostasis. We found that nitrating conditions after exposure to peroxynitrite strongly inhibited OASTL activity. Among OASTLs, OASA1 was markedly sensitive to nitration as demonstrated by the comparative analysis of OASTL activity in nitrated crude protein extracts from wild type and different oastl mutants. Furthermore, nitration assays on purified recombinant OASA1 protein led to 90% reduction of the activity due to inhibition of the enzyme, as no degradation of the protein occurred under these conditions. The reduced activity was due to nitration of the protein because selective scavenging of peroxynitrite with epicatechin impaired OASA1 nitration and the concomitant inhibition of OASTL activity. Inhibition of OASA1 activity upon nitration correlated with the identification of a modified OASA1 protein containing 3-nitroTyr302 residue. The essential role of the Tyr302 residue for the catalytic activity was further demonstrated by the loss of OASTL activity of a Y302A-mutated version of OASA1. Inhibition caused by Tyr302 nitration on OASA1 activity seems to be due to a drastically reduced O-acetylserine substrate binding to the nitrated protein, and also to reduced stabilization of the pyridoxal-5′-phosphate cofactor through hydrogen bonds. This is the first report identifying a Tyr nitration site of a plant protein with functional effect and the first post-translational modification identified in OASA1 enzyme.